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QM575  .K95  On  neurokeratin 


ON  NEUROKERATIN 
575 


\W    \Y.    KIHNE 

1'ltOFrseon   OK   FHTBIOLOaY   IS    llh'DK.I.i'.KKii    INlVKIt-MTY 

and  R.   II.  (IIITTENDEN 

PBORUOB  OK  PHYSIOLOGICAL  CfiXJdBTST   IN  YALE  T'NIVERSITT 


REPRINTED  FROM  THE  SEW   YOBE  MEDICAL  JOURXAI. 
FUR   FEliinWRY  ?:?  AND   MARCH  1,   1890 

COLUMBIA  UNIV'FRSITY 

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1).     A  PPL  ETON     AXD    COMPANY 
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ON  NEUROKERATIN 


By    \Y.    KUHNE 

PROFESSOR  OF   PHYSIOLOGY  IN    HEIDELBERG   UNIVERSITY 

and  R.   II.  CHITTENDEN 

PROFESSOR  OF   PUTSIOLOGICAL  CHEMISTRY    IN   YALE    UNIVERSITY 


EKPRIN1ED  FROM  THE  SEW   YOJBR  MEDICAL  ,/OCRXAL 
FOR  FEBRUARY  22  AND  MARCH  1,  1890 


N  E  W     YORK 

I).    APPLET  ON     AND    COMPANY 

1890 


Q  A!  5  75" 


ON  NEUROKERATIN.* 


Tiik  name  neurokeratin  f  was  applied  to  the  substance 
contained  in  mednllated  nerves  and  in  the  central  nerve 
organs  which  is  insoluble  in  alcohol  and  ether,  in  gastric 
and  pancreatic  juice,  and  in  dilute  caustic  potash.  It  is 
therefore  a  substance  characterized  by  extreme  insolubil- 
ity. <>nlv  the  tissues  and  organs  of  the  nervous  system 
and  the  horny  structures  of  the  epidermis  yield  such  an  in- 
soluble bodv,  so  resistant  to  the  action  of  all  solvents,  ex- 
cept such  as  are  powerful  decomposing  agents. 

The  preparation  of  neurokeratin  depends  upon  the  re- 
moval from  the  nerve  tissue  of  the  peculiar  histogenetic  sub- 
stances present  there,  such  as  albumin,  collagen,  elastin,  and 
nuclein,  likewise  upon  the  separation  of  all  fat  and  of  the 
so-called  myeline  substances — viz.,  protagon,  lecithin,  cere- 
brin,  cholesterin,  and  possibly  soaps,  and  also  of  the  ex- 
tractive matters  soluble  in  water  common  to  all  tissues. 

The  finding  of  neurokeratin  led  to  the  discovery  that 
the  nerve  medulla  was  more  complex  in  its  composition  than 
had  before  been  supposed,  especially  as  the  presence  of  pro- 
teid  matter  was  proved  at  the  same  time.     Furthermore,  it 

*  PuUlifshcd  also  in  the  Zeitsclirift  fur  Biologie,  Band  xxvi. 

f  A.  Ewald  and  \V.  Kiiline,  l*el>er  einen  neuen  Bestandtheil  des 
Xcrvoiisystcins,  Yerhandl.  d.  naturhist.  nicd.  Yereins  zu  Heidelberg, 
n.  V.,  Band  i,  1877,  p.  857. 


4  ON  NEUROKERATIN. 

was  likewise  ascertained  that,  in  addition  to  the  neurokera- 
tin of  the  medullary  sheath,  other  sheaths  of  similar  ma- 
terial were  also  present  in  the  medulla.  In  spite  of  these 
facts,  however,  we  shall  in  the  present  article,  for  the  sake 
of  retaining  simple  terms,  follow  the  old  use  of  the  word 
myeline  and  apply  to  the  bodies  extracted  by  alcohol,  ether, 
chloroform,  benzol,  or  carbon  disulphide  the  name  of  med- 
ullary or  myeline  substances,  although  the  term  is  now 
used  in  a  much  narrower  sense. 

In  order  to  obtain  neurokeratin  it  is  necessary  to  re- 
move the  medullary  substances  or  myelines  from  the  brain 
or  nerve  tissue,  as  well  as  to  free  the  latter  from  their  pe- 
culiar tissue  framework  by  digestion  and  from  nuclein  by 
extraction  with  alkali.  As  has  been  previously  stated  in 
another  place,  it  makes  no  difference  in  the  result  as  to  the 
order  of  treatment,  a  fact  of  considerable  importance,  since 
the  preparation  of  neurokeratin,  by  first  digesting  the  nerve 
tissue  and  then  extracting  the  myeline  substances,  furnishes 
proof  that  the  long  extraction  of  the  medullary  bodies  with 
alcohol,  etc.,  does  not  generate  the  insoluble  substance,  a 
fact  which  affords  evidence  of  the  pre-existence  of  neuro- 
keratin. 

For  the  microscopic  study  of  the  form  and  ai'range- 
ment  of  neurokeratin  the  method  of  treatment  commencing 
with  the  extraction  of  the  myeline  bodies  will  presumably 
remain  the  most  common,  although  even  for  this  purpose 
the  reversed  method  of  treatment  can  be  used  with  equally 
good  results ;  for  the  preparation  of  the  substance  in  large 
quantities,  however,  only  the  first  method  has  hitherto  been 
used.  We  have  for  our  present  purposes  employed  both 
methods,  and  in  the  course  of  our  experiments  have  found 
that  digestion  of  the  tissue  prior  to  extraction  of  the  mye- 
line substances,  even  where  large  masses  of  brain  are  taken, 
is  not  only  practicable,  but  possesses  certain  advantages. 


ON    NEUROKERATIN. 


Neurokeratin  from   Brain. 


The  material  best  adapted  for  use  i<  the  human  brain, 
mainly  on  account  of  its  large  size.  It  was  always  ob- 
tained from  an  early  dissected  cadaver  well  preserved  in 
the  cold,  and  was  always  Fresh  and  without  trace  of  putre- 
factive odor.  In  every  preparation  the  first  step  consisted 
in  the  separation  of  the  pia  mater  under  water,  so  as  to 
remove  as  far  as  possible  all  traces  of  Mood. 

1.  Preparation  by  Digestion  after  Extraction  of  the 
Medullary  Substance. — Two  brains  were  beaten  up  with 
4,800  c.  c.  of  absolute  alcohol,  the  mass  pressed  through  a 
sieve,  and,  after  standing  twenty-four  hours,  collected  in  a 
cloth  bag  or  filter  and  the  fluid  pressed  out.  The  residue 
was  rubbed  to  a  fine  paste  with  a  like  volume  of  fresh  alco- 
hol, and,  after  forty-eight  hours,  again  pressed  out  and 
washed  with  cold  ether.  The  mass  was  then  thorounhlv 
washed  with  hot  ether  in  an  extractor  with  inverted  con- 
denser for  twenty-four  hours,  after  which  it  was  dried  in 
the  air,  then  boiled  with  two  litres  of  absolute  alcohol, 
pressed  through  muslin  and  placed  in  a  special  extraction 
apparatus,  in  which  it  was  kept  in  contact  with  boiling 
alcohol  for  forty-eight  hours.  This  apparatus  was  so  ar- 
ranged that  with  the  help  of  a  sieve  the  mass  of  tissue  was 
at  Hist  sunk  deep  into  the  alcohol,  then  raised  nearer  the 
surface,  and  finally  suspended  above  the  boiling  alcohol,  so 
that  at  the  last  it  was  extracted  solely  by  the  warm  fluid 
Bowing  back  from  the  inverted  condenser.  After  thorough 
pressure,  the  mass,  freed  in  this  manner  from  the  medullary 
substances,  was  boiled  with  two  litres  of  water,  in  order  to 
completely  remove  the  adhering  alcohol  and  at  the  same 
time  to  render  the  connective  tissue  more  digestible.  It 
was  then  placed  in  five  litres  of  an  extremely  active  gastric 
juice  containing  0-4  per  cent,  hydrochloric  acid  and  warmed 


<5  ON  NEUROKERATIN. 

at  40°  C.  for  four  days,  after  which  it  was  thrown  on  a 
large  filter  connected  with  a  pump  and  thoroughly  washed 
with  water  after  sucking  out  all  of  the  filtrate.  The  slight 
acidity  of  the  remaining  tissue  was  neutralized  with  dilute 
alkali,  after  which  it  was  subjected  to  a  trypsin  digestion,  in 
which  one  litre  of  an  active  pancreatic  juice  was  employed, 
prepared  in  the  usual  manner  from  one  hundred  grammes 
of  dry  pancreas  and  containing  0*5  per  cent,  of  sodium 
carbonate.  After  five  days'  digestion  at  40°  C.  the  undi- 
gested residue  was  washed  with  water,  placed  in  two  litres  of 
five-per-cent.  sodinm  carbonate  solution,  and  boiled  for  half 
an  hour.  The  residue,  after  cooling,  was  filtered  off,  washed 
with  water,  and  placed  in  three  litres  of  0-5-per-cent.  caustic 
potash,  where  it  was  allowed  to  remain  for  forty-eight  hours 
at  medium  temperature,  with  frequent  stirring,  in  order  to 
dissolve  the  nuclein  present.  After  decantation,  filtration, 
and  washing  with  water,  the  mass  was  acidified  with  0*4- 
per-cent.  hydrochloric  acid  and  subjected  to  a  pepsin  diges- 
tion for  several  days  in  two  litres  of  an  active  gastric  juice. 

Tn  this  last  digestion  there  was  no  noticeable  shrinkage 
of  the  mass,  but,  after  washing  out  the  acid  with  water,  we 
found  that  hot  alcohol  still  dissolved  considerable  matter, 
accompanied  by  a  swelling  or  softening  of  the  substance,  in 
consequence  of  which  the  solution  filtered  very  slowly. 
This  admixture,  consisting  presumably  of  cerebrin,  was  re- 
moved to  better  advantage  by  repeated  boiling  with  alco- 
hol, to  which  a  little  glacial  acetic  acid  had  been  added. 
Thereupon  the  mass  was  treated  again  with  caustic  potash, 
and  this  time  for  two  days  with  a  solution  of  five  per  cent, 
strength,  after  which  it  was  washed  with  water,  then  with 
acetic  acid,  again  with  water,  and  finally  with  alcohol  and 
ether. 

The  preparation  obtained  in  this  manner,  on  being  tested 
with  pure  pepsin  and  trypsin,  was  found  to  be  entirely  free 


ON    NEUROKERATIN.  7 

from  substances  capable  of  furnishing  peptone,  also  free 
from  digestible  albumin?,  likewise  free  from  soluble  Lr(,l;i- 
tin,  since  tannic  acid  added  to  the  solution  obtained  l>\ 
boiling  with  water  gave  do  turbidity,  and  also  free  from 
nuolein,  since  five- per  cent,  caustic  potash  solution  dissolved 
nothing  precipitable  by  acetic  acid.  We  were  not  able, 
however,  to  completely  remove  all  of  the  myelitic  bodies, 
for  apparently  a  trace  of  cerebrin  still  remained.  Except 
in  the  preparation  of  small  quantities,  as  in  the  quantitative 
determinations  to  be  described  later,  we  have  not  been  able 
to  obtain  a  sample  of  neurokeratin  which  would  not  give 
up  some  matter  on  long-continued  boiling  with  alcohol, 
benzol,  or  chloroform,  the  fluid  on  evaporation  invariably 
leaving  a  slight  film  or  coat  on  the  glass  with  a  more  or  less 
fatty  feeling.  In  view  of  the  possibility  of  our  having  to 
deal  with  lime  or  magnesia  soaps  we  have  not  neglected,  as 
before  mentioned,  to  boil  with  alcohol  containing  either 
glacial  acetic  acid  or  hydrochloric  acid  without,  however, 
wholly  reaching  the  desired  end.  All  that  can  be  accom- 
plished is  to  have  the  neurokeratin  finally  brittle  in  hot 
alcohol,  so  that  the  latter  will  filter  easily  and  the  prepara- 
tion itself  not  feel  soft  or  fatty.  In  this  we  were  occasion- 
ally more  successful  with  the  neurokeratin  from  ox  brain 
than  from  human  brain,  hence  possibly  in  the  future  ox 
brain  will  be  the  preferable  material  to  work  with. 

2.  Preparation  by  Digestion  prior  to  Extraction  of  the 
Medullary  Substances. —  For  this  purpose  it  is  advantageous 
not  to  employ  more  than  one  brain  (about  1,300  grammes) 
in  a  single  preparation.  The  tissue  was  at  once  rubbed  up 
fine,  with  gradual  addition  of  0'4-per-cent.  hydrochloric 
acid,  and  to  this  was  finally  added  five  times  its  volume  of 
active  gastric  juice.  The  juice  was  always  prepared  by  the 
self-digestion  of  one  part  of  the  mucous  membrane  of  a 
pig's  stomach,  with  twenty  parts  of  0-4-percent,  hydrochloric 


g  ON  NEUROKERATIN. 

acid,  and  preserved  in  the  cold  after  being  moderately  thy- 
raolized.  As  a  rule,  the  brain-matter,  after  four  days'  di- 
gestion at  40°  C,  sank  so  completely  to  the  bottom  of  the 
vessel  that  the  excess  of  the  juice  could  be  decanted  off, 
while  the  residue  was  collected  on  a  filter,  again  rubbed  up 
as  fine  as  possible  in  a  porcelain  mortar,  and  once  more 
warmed  for  four  to  six  da\s  with  four  litres  of  fresh  gastric 
juice.  In  every  case  we  have  tested  a  sample  of  the  gastric 
juice  employed,  in  order  to  be  sure  of  its  activity,  by  try- 
ing its  solvent  power  on  blood-fibrin.  Although  the  diges- 
tion periods  were  quite  long,  we  never  observed  the  forma- 
tion of  any  mold  or  decomposition  through  the  growth  of 
micro-organisms,  though  the  clear  filtrates  frequently  broke 
down  on  long  standing  after  the  thymol  had  nearly  or  com- 
pletely left  them. 

After  the  second  digestion,  the  brain-matter  was  again 
collected  on  a  filter,  and,  after  being  washed,  was  either 
directly  freed  from  mveline  bodies  or  else  subjected  to  a 
trypsin  digestion  (possibly  unnecessary).  In  the  latter  case 
the  mass  was  neutralized  with  sodium-carbonate  solution, 
and  then  warmed  for  eight  days  at  40°  C.  with  one  litre  of 
well-thymolized  pancreatic  juice  (1  to  40)  containing  0*5  per 
cent,  of  sodium  carbonate.  At  the  end  of  the  digestion  the 
whole  mass  was  in  the  form  of  a  soapy  magma,  and,  as 
filtered  portions  on  neutralization  or  by  acidifying  were  not 
rendered  turbid,  the  entire  mixture  was  at  once  acidified 
with  hydrochloric  acid  up  to  about  0*1  per  cent.,  and  then 
^shaken  with  ether.  The  treatment  with  acid  was  necessary, 
as  the  ether  would  not  separate  well,  if  at  all,  from  the 
alkaline  fluid.  The  separation  of  lecithin,  protagon,  and 
cholestcrin  by  means  of  ether  was  accomplished  by  repeated 
shakings  in  a  large  separating  funnel,  and  at  the  same  time, 
also,  the  soluble  digestion  products  were  washed  out,  since 
the  brain-matter  under  the  ether  formed  such  a  compact 


on    NEUROKERATIN.  9 

layer.  Boating  on  the  acid  fluid,  thai  the  latter  only  Deeded 

to  be  drawn  off  and  the  operation  repeated  with  fresh  dilute 
acid  and  water.  When  tlii^  had  been  continued  until  a 
portion  of  the  aqueous  fluid  was  not  rendered  tnrbid  by  a 
large  excess  of  alcohol,  the  pasty  like  mass  under  the  ether 
was  drawn  off,  mixed  with  alcohol  until  it  could  be  easily 
filtered,  boiled  with  60-per-cent.  alcohol  for  half  an  hour, 
and  filtered  on  a  hot-water  funnel.  The  filtered  fluid,  at 
first  clear,  quickly  became  milky  on  cooling  from  the  sepa- 
rated cerebrin.  The  brain  residue  was  then  repeatedly 
boiled  with  absolute  alcohol,  benzol,  and  chloroform,  the 
filtrations  being  made  with  the  aid  of  the  hot-water  funnel. 
In  order  to  remove  the  nuclein  from  the  much-shrunken 
preparation,  it  was  treated  for  several  days  with  about  two 
litres  of  one-per-cent.  sodium  hydroxide  solution,  filtered, 
washed  with  the  alkali  until  the  washings  were  not  rendered 
turbid  by  acid,  then  with  dilute  hydrochloric  acid.  Finally, 
the  residue  was  boiled  with  alcohol  containing  a  little  hy- 
drochloric acid,  then  with  the  various  solvents  for  the  mye- 
line  bodies,  and  at  last  thoroughly  washed  with  ether. 

Whether  it  is  possible  by  either  one  of  the  above  meth- 
ods to  prepare  larger  quantities  of  neurokeratin  pure  is  un- 
certain. We  need  to  remember,  in  considering  the  prepara- 
tion of  this  substance,  that  we  are  dealing  with  a  body  which 
can  not  be  dissolved  unaltered  and  then  reprecipitated,  but 
with  one  which  can  only  be  obtained  as  a  residue,  and,  fur- 
ther, that,  in  spite  of  great  patience,  we  have  not  been  able, 
as  before  mentioned,  to  entirely  remove  the  myeline  bodies. 
A  further  difficulty  is  to  be  found  in  the  visible  admixture  of 
insoluble  pigments,  for  neurokeratin  obtained  from  thegraa 
substance,  or  from  the  entire  brain,  has  a  distinct  yellowish 
color,  while  that  prepared  from  the  white  substance  is  color- 
less. Another  slighl  impurity  is  to  be  found  in  the  presence 
of  paper  fibers,  although  we  have  avoided  these  as  much  as 


10  ON   NEUROKERATIN. 

possible,  even  at  the  expense  of  our  product,  by  leaving 
considerable  of  the  sticky  substance  adhering  to  the  paper. 
This  we  further  endeavored  to  reduce  to  a  small  micro- 
scopic residue  by  working  the  preparation,  while  still  moist 
with  alcohol  and  ether,  on  a  glass  plate  with  a  blunt  knife, 
in  order  to  felt  the  fibers  into  little  flocks,  many  of  which 
tended  to  stick  to  the  blade.  Still  more  was  removed,  after 
drying  and  pulverizing  the  product,  by  sifting  the  powder 
through  fine  mull.  We  have  also  tried  shaking  and  wash- 
ing the  substance  with  heavy  fluids,  such  as  chloroform  or 
carbon  disulphide,  but  without  result. 

The  following  analyses  were  made  with  products  dried 
at  110°  C.  until  of  constant  weight.  Carbon  and  hydrogen 
were  determined  by  combustion  in  oxygen  gas  in  a  tube 
filled  with  granular  oxide  of  copper,  with  an  anterior  layer 
of  chromate  of  lead  and  a  roll  of  reduced  copper.  Nitro- 
gen was  determined  as  gas  * ;  sulphur,  according  to  Ham- 
marsten's  method. 

I.  Neurokeratin  from  two  human  brains,  in  which  the 
medullary  substances  were  first  extracted,  using  only  alcohol 
and  ether,  and  the  residue  of  tissue  then  digested  : 

I.  0*5377  gramme  of  substance  gave  0'3522  gramme  H20  =  7*27 

per  cent.  H,  and  1*0928  gramme  C02  =  55*42  per  cent.  C. 
II.  0-4389  gramme  of  substance  gave  0*2850  gramme  H20  =  7*21 
per  cent.  H,  and  0*8926  gramme  C02  =  55*46  per  cent.  C. 

III.  0*5250  gramme  of  substance  gave  63*7  c.  c.  N  at  19*6°  C.  and 

754*9  mm.  pressure  =  14*13  per  cent.  N. 

IV.  0 '52*78  gramme  of  substance  gave  63*7  c.  c.  N  at  19*6°  C.  and 

761*4  mm.  pressure  =  14*18  per  cent.  N. 
V.  0*6852  gramme  of  substance,  fused  with  KOH  +  KN03,  gave 
»  0*0930  gramme  BaS04  =  1*86  per  cent.  S. 

VI.  0*5455  gramme  of  substance,  fused  with  KOH  +  KN03,  gave 

0*0744  gramme  BaS04  —  1  *87  per  cent.  S. 
VII.  0*6597  gramme  of  substance  gave  0*0075  gramme  ash  (calcium 

phosphate)  =  1*13  per  cent. 

*  Zeitschrift  fur  Biologie,  Bd.  xix,  p.  166. 


ON    M'.l  i;uKi;i;.\TIN.  \{ 

VIII.   0*7247    grain n f  sill >st ati<<-  gave   ihiil'.i:!   gramme   ash  =  L'28 

per  (rut. 

Ptrcentagt  Composition  <>/  tin-  Ash-free  Substance, 

\  range, 
(' 50  09         5613  56 -I  I 


II 7'88  7-30  

N 14:50         14-35         

S 1ST  1 

O 


. .  733 

.  .  14-32 

88  1-H8 

.  .  20-36 

ioo-oo 


II.  As  preparation  No.  1  had  a  somewhat  greasv  feel- 
ing and  yielded  a  little  something  to  hot  benzol,  it  was 
again  thoroughly  extracted  with  boiling  benzol,  chloroform, 
alcohol,  and  carbon  disulphide,  and  an  endeavor  made  to 
free  it  still  more  completely  from  paper  fibers.  After  being 
dried  at  110°  C.,it  was  analyzed  with  the  following  results: 

I.  0*3817  gramme  of  substance  gave  0-2468  gramme  HaO  =  7'18 
per  cent.  H,  and  0-7830  gramme  C0a  =  5594  per  cent.  C. 
II.  03825  gramme  of  substance  gave  0*2480  gramme  H3()  =  7*28 
per  cent.  H,  and  0-7806  gramme  COa  =  5565  per  cent.  C. 

III.  03724  gramme  of  substance  gave  0-2416  gramme  H20  —  7*20 

per  cent.  H,  and  0*7620  gramme  C02  =  55-79  per  cent  C. 

IV.  0-3154  gramme  of  substance  gave  35-9  c.  c.  N  at  5  4°  C.  and 

7543  mm.  pressure  =  1391  per  cent.  N. 
V.  0-398.")  gramme  of  substance  gave  45*4  c.  c.  N  at  6-0°  C.  and 

757'6  mm.  pressure  =  13-95  per  cent.  N. 
VI.  05 126  gramme  of  substance,  fused  with   KOII  +  KX0S,  gave 

00612  gramme  BaS0«  =  1-63  per  cent.  S. 
VII.  0-4987  gramme  of  substance,  fused  with   KOII  +  K\03,  gave 
00570  gramme  BaS0«  =  1'57  per  cent.  S. 
VIII.  0'5726  gramme  of  substance  gave  0-0050  gramme  ash  =  0-87 
per  cent. 
IX.  0-6166  gramme  of  substance  gave  0-0057  gramme  ash  =  o-92 

per  cent. 
X.  0-4985  gramme  of  substance,  fused  with  KOII  4-  KN'O,,  precipi- 
tated with  molybdio  solution,  and  finally  with  magnesia  mixt- 


c 

56-44 

56-15 

56-29 

H 

'7-24 

V-29 

1-26 

N 

S 

0  .... 

12  ON  NEUROKERATIN. 

ure,  gave  0-0041  gramme  Mg2P207  =  0-22  per  cent.  P,  or  no 
more  than  the  phosphorus  of  the  calcium  phosphate  of  which 
the  ash  was  composed. 

Percentage  Composition  of  the  Ash-free  Neurokeratin. 

Average. 

56-29 

....  7-26 

14-04     14-08      14-06 

1-66       1-60  1-63 

20-76 

100-00 

As  is  seen  from  the  results,  the  more  complete  extraction 
of  the  cerebrin  (?)  makes  no  special  difference  in  the  per- 
centage of  composition  when  contrasted  with  No.  1. 

III.  Neurokeratin  from  the  brain  of  a  child  eighteen 
months  old,  purified  as  in  No.  II;  first  digested  and  then 
the  medullary  substances  extracted  : 

I.  0-3854  gramme  of  substance  gave  0-2580  gramme  H20  =  7'43 

per  cent.  H,  and  0-7894  gramme  C02  =  55'85  per  cent.  C. 
II.  0-3660  gramme  of  substance  gave  0-2447  gramme  H20  =  7'42 
per  cent.  H,  and  0-7524  gramme  C02  =  56'05  per  cent.  C. 

III.  03474  gramme  of  substance  gave  36-2  c.  c.  N  at  5'4°  C.  and 

755'7  mm.  pressure  =  12-76  per  cent.  N. 

IV.  0-3414  gramme  of  substance  gave  36-2  c.  c.  N  at  6'4°  C.  and 

755-0  mm.  pressure  =  12-92  per  cent.  N. 
V.  0-4923  gramme  of  substance,  fused  with  KOH  +  KN03,  gave 

0-0610  gramme  BaS04  =  1'70  per  cent.  S. 
VI.  0-5807  gramme  of  substance,  fused  with  KOH  +  KN03,  gave 

0-0743  gramme  BaS04  =  1  75  per  cent.  S. 
VII.  0-4897  gramme  of  substance  gave  0-0076  gramme  ash  =  l-57 
per  cent. 
VIII.  0-4784  gramme  of  substance  gave  0-0073  gramme  ash  =  1*53 
per  cent. 
IX.  0-4923  gramme  of  substance,  fused  with  KOH  +  KN03,  precipi- 
tated with  molybdic  solution,  and  finally  with  magnesia  mixt- 
ure, gave  0-0079  gramme  Mg2P207  =  0-44  per  cent.  P,  corre- 
sponding to  the  calcium  phosphate  of  the  ash. 


ON   NEUROKERATIN.  13 

I'rr<-rn/ii(/)    CiiiiijMixitinn    •>(  tin     Ash-l'm    Su/ixtmirr. 

Average. 
C 5672         6«'92         56-82 

II 755           7-54         7-54 

N 12-98        18-12         18-04 

S 17:;           1-78  175 

<> 20-85 

lUIIMHI 

IV.   Neurokeratin  from  tlie  brain  of  an  adult;  method 
of  preparation  same  as  in  No.  Ill  : 

I.  0'4082  gramme  of  substance  gave  02930  gramme  H30  =  7'97 

per  cent.  II,  and  0-8688  gramme  C0a  =  58  04  per  cent.  0. 
II.  0  157-  gramme  of  substance  gave  0*8276  gramme  H20  =  7'95 
per  cent.  H,  and  09725  gramme  C02  =  58-00  per  cent.  C. 

III.  0*3149  gramme  of  substance  gave  292  c.  c.  N  at  4'6°  C.  and 

7526  mm.  pressure  =  11  34  per  cent.  N. 

IV.  0-3856  gramme  of  substance  gave  35-8  c.  c.   N  at  5-6°  C.  and 

759-9  mm.  pressure  =  11  42  per  cent.  N. 
V.  0*6109  gramme  of  substance,  fused  with   KOH  +  KX03,  gave 

0-0672  gramme  BaS0<  =  1*80  per  cent.  S. 
VI.  0*8663   gramme  of  substance,  fused   with   KOH  +  KN03,  gave 

0-0490  gramme  BaS04  =  1'89  per  cent.  S. 
VII.  0*8662  gramme  of  substance,   fused  with   KOH  -f  KX03,  gave 

0*0482  gramme  BaS04  =  1-86  per  cent.  S. 
VIII.  0*4853  gramme  of  substance  gave  0'0036  gramme  ash  =  074 
per  cent. 
IX.  0-9788  gramme  of  substance  gave  00073  gramme  ash  =  0*74  per 

cent. 
X.  0-5108  gramme  of  substance,  fused  with  KOH  +  KX03,  precipi- 
tatfd  with  molvbdic  solution,  and  finally  with  magnesia  mixt- 
ure, gave  0*0022  gramme  MggPgOi  =0*12  per  cent.  P,  corre- 
sponding to  the  phosphate  of  the  ash. 

I 'it-rentage  Composition  of  ili<-  Ash-free  Substance, 

Average. 
C 58-47     58-43 58-45 

11 8-03       801       8*02 

N 11*42  11*60  11-46 

S L-88   1-91   1:89  1-87 

0 20  20 

100-00 


14 


ON   NEUROKERATIN. 


V.  Neurokeratin  from  the  brain  of  a  man  twenty-one 
years  old ;  method  of  preparation  same  as  in  Nos.  Ill  and 
IV: 

I.  0-5783  gramme  of  substance  gave  0-3803  gramme  H20  =  7'30 

per  cent.  H,  and  T1820  gramme  C02  =  55-73  per  cent.  C. 
II.  0-3695  gramme  of  substance  gave  0-2463  gramme  H20  =  7'40 
per  cent.  H,  and  0-7606  gramme  C02  =  56-13  per  ceDt.  C. 

III.  0"3881  gramme  of  substance  gave  0-2578  gramme  H20  =  7'38 

per  cent.  H,  and  0-7965  gramme  C02  =  55-96  per  cent.  C. 

IV.  0-4720  gramme  of  substance  gave  48-l  c.  c.   N  at  5*0°  C.  and 

755-8  mm.  pressure  —  12-50  per  cent.  N. 
V.  0*8833  gramme  of  substance  gave  39-9  c.  c.  N  at  6*4°  C.  and 

756-3  mm.  pressure  =  12-71  per  cent.  N. 
VI.  0-5160  gramme  of  substance,  fused  with  KOH  +  KN03,  gave 

0-0825  gramme  BaS04  =  2~19  per  cent.  S. 
VII.  0-4806  gramme  of  substance,  fused  with  KOH  +  KN03,  gave 
0-0768  gramme  BaS04  =  2-19  per  cent.  S. 
VIII.  0-5401  gramme  of  substance  gave  0-0130  gramme  ash  =  2-40 
per  cent. 
IX.  0-2742  gramme  of  substance  gave  0-0065  gramme  ash  =  2-37 

per  cent. 
X.  0-4805  gramme  of  substance,  fused  with  KOH  +  KN03,  precipi- 
tated with  molybdic  solution,  and  finally  with  magnesia  mixt- 
ure, gave  0-0056  gramme  Mg2P207  =  0-32  per  cent.  P,  corre- 
sponding to  the  calcium  phosphate  of  the  ash. 


Percentage  Composition  of  the  Ash-free  Substance. 


c 

. . .     57-09 

57-48 

57-32 

H 

7-48 

7-58 

7-56 

S 

0  .... 

12-80     13-00 


2-i 


Average. 
.     57-27 

7-54 
.      12-90 

2-24 
20-03 


24 


I. 

C 56-11 

H 733 

N 14-32 

S 1-88 


100-00 

Neurokeratin. 

Hair- 

II. 

III. 

IV. 

v. 

keratin. 

56-29 

56-82 

58-45 

57-29 

49-45 

7-26 

7-54 

8-02 

7-54 

6-52 

14-06 

13  04 

11-46 

12-90 

16-81 

1-63 

1-75 

1-87 

2-24 

4-02 

Ash 


1-21 


0-89 


1-55 


074 


2-38 


1-01 


ON   NEUROKERATIN.  15 

Examination  of  the  above  given  results  shows  many 
points  of  interest  in  spite  of  the  individual  variations,  which 
were  perhaps  to  be  expected  from  the  manner  of  prepara- 
tion. First  of  all  we  notice  the  absence  of  phosphorus,  con- 
cerning which  we  have  reported  in  detail,  because  it  mighl 
naturally  he  expected  that  an  indigestible  body  like  neuro- 
keratin should  contain  some  substance  related  to  the  nu- 
cleins,  as,  for  example,  a  nuclein  insoluble  in  caustic  alka- 
lies. The  abseuce  of  phosphorus,  however,  shows  that  such 
is  not  the  case.  Secondly,  the  low  amount  of  sulphur  is  in- 
teresting, especially  when  compared  with  older  data,  which 
show  2'93  per  cent,  in  the  case  of  neurokeratin  from  the 
brains  of  oxen  and  in  preparations  which  had  been  extracted 
with  only  0*5-per-cent.  caustic  soda.  In  Nos.  I  and  II 
the  preparation  was,  to  be  sure,  extracted  with  an  alkali  of 
ten  times  the  above  concentration,  but  of  the  three  other 
specimens,  all  of  which  were  treated  with  only  one-per-cent. 
caustic  alkali,  Xos.  Ill  and  IV  do  not  show  any  notice- 
ably higher  percentage.  In  preparation  No.  V,  however, 
the  amount  of  sulphur  rises  to  2'24  per  cent.,  although  it 
was  prepared  in  exactly  the  same  manner  as  the  preceding 
preparations  and  with  no  less  care ;  still  the  higher  amount 
of  ash  is  strongly  suggestive  of  some  impurity.  As  to  how 
much  sulphur  is  to  be  attributed  to  the  albumins  and  albu- 
minoids is  obviously  a  difficult  question,  but  no  more  diffi- 
cult to  decide  than  in  the  case  of  keratin  and  neurokeratin, 
since  both  continuously  give  up  sulphur  to  caustic  alkali, 
and  that  without  losing  the  lead  reaction  of  the  more  loosely 
combined  sulphur.  Doubtless  the  percentage  of  residual 
sulphur  in  these  bodies  is  more  dependent  upon  the  abso- 
lute amount  of  alkali  employed,  upon  the  temperature  or 
upon  the  duration  of  the  extraction,  than  upon  the  concen- 
tration of  the  fluid. 

As  preparations  I  and  II  show  a  difference  of  0'25  per 


IQ  ON   NEUROKERATIN. 

cent,  of  sulphur,  we  endeavored  to  ascertain  whether  the 
fluids  used  in  the  extraction  of  the  myeline  bodies  contained 
any  sulphur  at  the  end  of  the  process.  On  testing  the  resi- 
dues, however,  with  acetate  of  lead  and  potassium  hydroxide, 
we  failed  to  find  any  sulphur  reaction.  Preparation  No.  IT, 
which  was  more'  thoroughly  extracted  with  alcohol,  ben- 
zol, etc.,  was,  however,  0*32  per  cent,  poorer  in  ash  than 
No.  I. 

Of  the  other  constituents  of  neurokeratin,  carbon  is  some- 
what higher  and  nitrogen  somewhat  lower  than  the  percent- 
ages contained  in  albuminous  bodies. 

In  this  connection  it  seemed  very  desirable  to  compare 
the  composition  of  ordinary  keratin  with  that  of  neurokera- 
tin, and,  as  the  horny  substance  of  the  epidermis  had  never 
been  studied,  when  purified  from  albumin  by  the  method 
employed  by  us  for  neurokeratin,  we  have  subjected  hair  to 
this  process,  purifying  it  by  digestion. 

With  this  end  in  view  a  quantity  of  white  hair  from  rab- 
bits, weighing  500  grammes,  was  digested  for  a  long  time 
with  gastric  juice  and  afterward  with  pancreatic  juice.  The 
quantity  did  not  apparently  diminish  any  by  this  treatment, 
although  the  gastric  juice  became  somewhat  colored  and  the 
fluid  gave  a  not  inconsiderable  flocculent  turbidity  on  neu- 
tralization. After  thorough  washing  with  water,  the  moist 
and  matted  mass  was  next  treated  for  twenty-four  hours  with 
seven  litres  of  0*5-per-cent.  caustic-soda  solution,  after  which 
it  was  collected  on  a  cloth  filter  and  thoroughly  washed  with 
cold  and  hot  water.  On  testing  a  sample,  it  was  found  to 
still  give  up  considerable  sulphur  to  alkalies,  consequently 
the  extraction  with  alkali  was  repeated  and  in  this  case  con- 
tinued for  three  days  with  five-per-cent.  caustic-potash  so- 
lution. The  mixture  was  then  greatly  diluted  with  water, 
filtered  through  flannel,  the  residue  washed  with  water,  and 
lastly  with  0*5  per  cent,  hydrochloric  acid.    The  hair  while 


ON    NEUROKERATIN.  17 

in  the  alkali  appeared  < j u it e  yellow  in  color,  but  after  the 
washing  it  became  glistening  white  again,  and,  as  il  was  now 
somewhat  broken  to  pieces  and  evidently  more  brittle  than 
before,  we  thought  it  advisable  while  in  this  condition  to 
extract  it  once  more  very  thoroughly  with  gastric  juice. 
Much  to  our  astonishment,  however,  we  found,  after  warm- 
ing the  mass  with  ten  litres  of  gastric  juice  for  twenty -four 
hours,  that  full  four  fifths  of  the  substance  had  disappeared; 
on  treating  the  residue,  however,  with  fresh  gastric  juice,  it 
was  not  visibly  altered.  Under  the  microscope  the  hair 
structure  was  still  visible,  although  the  pieces  were  very  soft, 
somewhat  swollen  and  pale,  and  fell  apart  readily  into  single 
flakes  or  scales.  The  mass  was  finally  boiled  with  alcohol, 
washed  with  ether,  and  dried  at  110°  C.  The  following  re- 
sults show  its  composition  : 

White  rabbits'  hair,  purified  by  digestion,  by  removal  of 
the  fat  and  by  caustic  soda  : 

I.  0*5348  gramme  of  substance  gave  0*3120  gramme  H20  =  6*48 

per  cent.  H,  and  0-9616  gramme  COQ  =  4903  per  cent.  C. 
II.  03162  gramme  of  substance  gave  0-1832  gramme 'H20  =  6-43 
per  cent.  II,  and  0-6655  gramme  C02  =  48-77  per  cent.  C. 

III.  0-5340  gramme  of  substance  gave  0-3115  gramme  H20  =  647 

per  cent.  H,  and  0*9605  gramme  C02  =  4905  per  cent.  C. 

IV.  04603  gramme  of  substance  gave  615  c.  c.  N  at  56°  C.  and 

763*8  mm.  pressure  =  1653  per  cent.  X. 
V.  0*5312  gramme  of  substance  gave  72-6  c.  c.  N  at  5*6°  C.  and 

757*9  mm.  pressure  =  1677  per  cent.  X. 
VI.  05300  gramme  of  substance  gave  71  "8  c.  c.  X  at  56°  C.  and 

7  "'7  9  mm.  pressure  =  16'62  per  cent.  X. 
VII.  0-5889  gramme  of   substance,  fused  with  KOII  +  KNOj,  gave 

0*1712  gramme  BaS04  =  3-99  per  cent.  S. 
VIII.  0-6805  gramme  of  substance,  fused   with   KOII  +  KX03,  gave 
0-1963  gramme  BaSO«  =  396  per  cent.  S. 
IX.  0-6919  gramme  of  substance  gave  0*0072  gramme  ash  =  1*04  per 

cent. 
X.  0'5921  gramme  of  substance  gave  0-oo.v.)  gramme  a>li  =  099  per 
cent. ;  average  I  01  per  cent,  ash,  with  much  silica. 


18  ON   NEUROKERATIN. 

Percentage  Composition  of  the  Ash-free  Substance. 

Average. 

C 49-53  49-27  49-55  49  45 

H. .  . .   6-54   6-50   6-53   6'52 

N 16-70    16-94    16-79 16-81 

S 4-03      4-00  4-02 

0 23-20 


100-00 
We  do  not  care  at  the  present  time  to  mate  any  close 
comparison  of  these  results  with  the  analyses  of  keratin  pre- 
viously recorded,  and  will  only  call  attention  to  the  fact  that 
our  results  do  not  differ  essentially  from  the  older  analyses 
of  keratin  from  hair,  except  that  our  figures  for  carbon  and 
nitrogen  are  somewhat  lower;  on  the  other  hand,  the  dif- 
ferences between  our  keratin  results  and  those  for  neuro- 
keratin are  very  great,  especially  in  the  case  of  sulphur,  which 
in  the  hair  amounts  to  four  per  cent,  or  more,  and  that  too 
in  spite  of  the  energetic  extraction  with  five-per-cent.  caustic 
potash.  In  making  a  comparison,  however,  we  need  hardly 
consider  the  sulphur,  which  certainly,  in  part  at  least,  comes 
from  some  admixed  body,  for  the. differences  between  the 
carbon  and  nitrogen  are  such  as  to  plainly  show  that  hair 
keratin  and  neurokeratin  can  not  be  directly  grouped  to- 
gether chemically.  Indeed,  the  above-stated  analyses  con- 
stitute a  most  urgent  reason  for  considering  anew  the  whole 
keratin  question  in  order  to  clear  up  first  of  all  the  great  dif- 
erences  in  composition  between  the  different  keratins  from 
the  epidermis,  nails,  horn,  feathers,  etc. — differences  which 
have  long  been  known  to  exist. 

The  earlier  statements  regarding  the  comparative  in- 
solubility of  neurokeratin  from  ox  brain  we  have  been  able 
to  substantiate  with  neurokeratin  from  the  human  brain; 
for,  while  rabbits'  hair  placed  in  five-per-cent.  caustic  potash 
for  four  weeks  entirely  disappeared,,  neurokeratin  was 
hardly  altered  by   this  treatment,  and,  further,  on  boiling 


ON  NEUROKERATIN.  19 

with  dilute  sulphuric  acid,  it  left  :i  much  larger  residue  than 
did  hair. 

In  attempting  to  make  a  comparative  determination  of 

the  absolute  and  relative  amounts  of  tyrosine  and  leucine 
which  could  he  obtained  from  neurokeratin  and  from  hair 
we  met  with  a  somewhat  surprising  result.  Neurokeratin, 
on  being  boiled  with  dilute  sulphuric  acid  for  thirty-six  to 
forty-eight  hours,  \  ielded  both  bodies  in  abundance,  the 
tyrosine  predominating,  as  is  also  the  case  with  ox  horn 
when  purified  in  the  ordinary  way  or  by  digestion.  With 
the  purified  rabbit's  hair,  on  the  other  hand  (purified  by 
digestion),  we  were  not  able  with  five  grammes,  a  quan- 
tity ordinarily  more  than  sufficient,  to  obtain  any  trace  of 
tyrosine  whatever.  There  was  an  abundance  of  leucine,  hut 
no  tyrosine;  even  the  fluid  from  which  the  tyrosine  should 
have  crystallized,  if  present,  gave  only  a  slight  orange 
color  on  being  tested  by  the  Millon-IIoffmann  test.  White 
rabbit's  hair,  unpurified  or  simply  purified  by  digestion, 
yielded  tyrosine  quite  plainly — apparently  less,  however,  than 
yielded  by  the  same  quantity  of  horn,  and  even  less  than 
from  fibrin.  On  testing  the  material  furnished  by  a  hair 
dealer,  from  which  our  purified  preparation  was  made,  five 
grammes  boiled  with  dilute  acid  failed  to  give  hardly  any 
better  reaction  for  tyrosine  than  the  purified  product,  and  it 
was  only  by  taking  fifteen  grammes  of  the  material  that  we 
were  able  to  obtain  suffieiem  tyrosine  to  recognize  the  crys- 
tals. It  seems  probable,  therefore,  in  spite  of  the  protesta- 
tions of  the  dealer  to  the  contrary,  that  in  the  technical 
working  of  the  hair  some  powerful  agents  must  have  been 
used,  which  for  our  purpose  was  certainly  unfort:  nate. 

In  connection  with  our  study  of  this  insoluble  and  indi- 
gestible constituent  of  the  nervous  system,  we  have  tried  an 
experiment  of  some  morphological  interest  with  the  cord  of 
the  lobster.      We  dissected   out  as  clean    as   possible   tin- 


20  ON  NEUKOKERATIN. 

cords,  consisting  of  nerve  fibers  and  ganglia,  from  several 
medium-sized  lobsters,  an  operation  of  no  great  difficulty, 
and  digested  tbem  directly  with  gastric  juice  and  then  with 
pancreatic  juice,  after  which  the  more  or  less  adherent  resi- 
due was  extracted  with  alcohol  and  ether,  and  finally  with 
caustic  soda.  That  the  residue  consisted  of  chitin  and  not 
of  neurokeratin  was  shown  by  dissolving  it  in  concentrated 
sulphuric  acid,  pouring  the  solution  into  boiling  water, 
neutralizing,  and  testing  its  reducing  action  on  alkaline 
copper  solution.  It  gave  a  strong  reduction,  similar  to 
grape  sugar. 

Quantitative  Determination  of  Neurokeratin. — The  meth- 
od employed-  for  the  separation  and  purification  of  this 
body  can  also  be  used  in  its  quantitative  determination. 
Aside  from  the  long  waiting,  the  process  can  be  carried  out 
quite  simply  and  accurately,  but  it  is  naturally  a  somewhat 
tedious  one.  Only  one  filter  needs  to  be  used — viz.,  that 
upon  which  the  substance  is  finally  weighed — and,  aside  from 
the  watch-glass  upon  which  the  original  tissue  is  weighed, 
only  one  vessel  is,  as  a  rule,  needed  in  the  process.  The 
method,  however,  needs  to  be  modified  somewhat  in  the 
case  of  the  white  substance  of  the  brain,  and  also  when  ex- 
traction of  the  myeline  bodies  precedes  the  digestion. 

We  have  confined  our  determinations  wholly  to  the 
nerves  and  brain  of  man  ;  both  should  be  freed  as  much  as 
possible,  before  weighing,  from  external  connective  tissue 
and  vessels,  and  also  be  cut  into  small  pieces  with  scissors. 
As  in  this  process,  and  in  the  separation  of  the  gray  and 
white  substance,  it  is  impossible  to  avoid  the  loss  of  water 
by  evaporation,  and  as  the  material  should  amount  to  about 
fifty  grammes,  the  preliminary  weight  can  seldom  be  exact. 
We  have,  however,  weighed  each  quantity  as  rapidly  and 
accurately  as  possible,  ordinarily  placing  the  greatest  value 
on  the  final  weighing  of  the  prepared  substance.     In  this 


OX    NEUROKERATIN.  21 

way  the  different  determinations  were  made  under  exactly 
the  same  conditions,  ami  are  hence  Btrictly  comparable. 

Nerves  or  portions  of  the  gray  brain  substance  were 
rinsed,  with  five  to  >i\  times  their  quantity  of  gastric  juice, 
into  a  glass-stoppered  separating  funnel  of  Too  to  900  c.c. 
capacity,  and  subjected  in  this  vessel,  with  frequent  vigorous 
shaking,  to  digestion  at  40°  C.  for  eight  to  fourteen  days. 
By  thi>  treatment  the  mass  became  a^  finely  divided  as  if  it 
had  been  rubbed  up  in  a  mortar.  In  the  case  of  the  white 
substance  of  the  corpus  callosum,  however,  this  treatment  is 
not  so  successful,  since  the  substance  sticks  in  part  so  closely 
to  the  sides  of  the  vessel  above  the  fluid  that  it  is  more  or 
less  withdrawn  from  the  digestive  juice.  In  this  case,  there- 
fore, the  material  was  rubbed  quite  tine  with  the  gastric  juice, 
and  the  digestion  carried  on  in  a  beaker,  and  when  thesub- 
Btance  had  settled  to  the  bottom  of  the  dish,  which  gener- 
ally occurred  on  the  fourth  day,  the  clear  supernatant  fluid 
H;h  in  great  part  poured  off  and  the  sediment  again  rubbed 
up  with  a  new  lot  of  fresh  gastric  juice.  After  the  pepsin 
digestion  was  finished,  the  residue  was,  at  the  beginning  of 
our  work,  subjected  to  the  action  of  trypsin,  a  step  which 
we  later  found  to  be  unnecessary. 

For  this  purpose  the  thick,  but  more  or  less  fluid,  residue 
was  neutralized  with  a  moderately  strong  solution  of  sodium 
carbonate,  and  then  treated  with  50  c.  c.  of  an  artificial  pan- 
creatic juice  for  eight  days,  at  40°  C,  after  which  it  was 
made  distinctly  acid  with  hydrochloric  acid  and  shaken 
with  ether.  As  a  rule,  we  were  generally  successful  in 
drawing  off  the  acid  watery  solution  beneath  the  layer  of 
undigested  matter,  cpiite  clear,  and  usually  also  the  wash 
fluids,  which  were  composed  of  0*4-per-cent.  hydrochloric 
acid.  If,  however,  the  fluids  did  show  a  DOticeable  tur- 
bidity, they  weir  passed  through  the  weighed  filter,  while 
the  main  bulk  of  the  undigested  matter  was  -till  kept  in  the 


22  ON   NEUROKERATIN. 

separating  funnel.  After  being  thoroughly  washed,  the 
residue  of  tissue,  made  into  a  magma  with  a  little  water,  was 
united  with  the  upper  ethereal  layer  by  the  addition  of  al- 
cohol, and  the  now  homogeneous  fluid,  together  with  the 
undissolved  matter,  was  allowed  to  flow  upon  the  filter 
which  was  placed  in  a  hot-water  funnel.  . 

In  order  to  avoid  all  loss,  the  separating  funnel  below 
the  stop-cock  must  be  cut  quite  short,  so  that  the  mouth  of 
the  funnel  can  be  well  rinsed  by  means  of  a  wash  bottle. 
The  hot-water  funnel,  on  account  of  danger  from  fire,  had 
attached  to  it  a  long  rubber  tube,  through  which  the  alco- 
hol passed  into  a  receptacle  placed  some  distance  away; 
Further,  in  order  to  facilitate  the  boiling  of  the  fluid  on  the 
filter,  the  rubber  tube  was  provided  with  a  spring  clip, 
which  could  be  closed  during  the  operation  of  boiling. 
After  the  substance  had  been  thoroughly  extracted  with 
alcohol  and  was  ready  to  be  washed  with  ether,  benzol,  or 
carbon  disulphide,  the  burner  was  naturally  extinguished 
and  the  tube  removed,  likewise  after  extraction  with  chloro- 
form, which  we  have  also  frequently  used.  The  various 
fluids  were  poured  upon  the  substance  boiling  hot,  while 
the  warm-water  funnel  was  still  hot,  and,  from  its  large 
size,  remained  so  for  some  time. 

With  this  arrangement  it  was  comparatively  easy  to 
free  the  digestion  residue  completely  from  recognizable 
traces  of  myeline  bodies — or,  in  other  words,  by  this  meth- 
od we  were  able  to  accomplish  what  we  did  not  succeed  in 
doing  with  larger  amounts  of  neurokeratin.  After  cooling, 
the  mass  was  treated  for  at  least  two  days  with  one  to  two 
litres  of  one-per-cent.  caustic  soda  for  the  removal  of  the 
nuclein.  The  alkali  was  allowed  to  flow  over  the  residue 
on  the  filter,  being  poured  back  and  forth  until  the  nuclein 
was  wholly  extracted  ;  it  was  then  washed  with  water,  after- 
ward with  dilute  hydrochloric  acid,  again  with  water,  both 


ON    NEUROKERATIN.  23 

cold  and  li<>t,  and  finally  with  alcohol  and  ether,  after  which 
it  was  dried    on    the    tiller,  first    in    t  he  air,  t  hen  at   110°  C, 

and  weighed. 

These  various  determinations  should  convince  those  who 

do  not  now  apparently  believe  in  the  coexistence  within 
the  nervous  system  of  the  myelitic  bodies  and  of  a  substance 
resistanl  to  the  action  of  the  digestive  fluids  and  of  caustic 
alkalies  that  neurokeratin  really  does  exist;  for  in  the 
weighed  residues  from  the  nerve  filters,  in  spite  of  all  that 
they  had  passed  through,  we  could  plainly  detect  micro- 
scopically the  hornv  sheaths  and  the  spongedike  neuro- 
keratin in  the  peculiar  knotty  or  gnarled  structure  of  the 
fragments  as  well  as  in  the  separated  short  cylinders  occa- 
sionally seen,  in  which  might  also  be  discovered  a  second 
still  smaller  cylinder  of  the  inner  horny  sheath.  The  frag- 
ments were  colored  intensely  blue  by  hematoxylin,  while 
by  Ileidenhain's  coloring,  with  the  supplementary  action 
of  chromates,  they  were  stained  a  blackish  blue.  They 
likewise  gave  the  lead  reaction  for  sulphur. 

I.  25  grammes  from  both  brachial  plexites  of  a  woman  seventy-two 

years  old  gave  0"079  gramme  of  neurokeratin  =  CK316  per  cent. 

II.  50  grammes  of  the  cortex  of  the  cerebellum  of  a  man  twenty-one 

years  old  pave  0-1560  gramme  of  neurokeratin  =  0-312  per  cent. 

III.  50  grammes  of  white  substance  (not  quite  free  from  gray)  from 

the  cerebrum  of  the  same  man  gave  T1217  gramme  of  neuro- 
keratin =  2243  per  cent. 

IV.  50  grammes  of  the  pure  white  substance  from  the  corpus  callosum 

of  a  man  fifty-seven  years  old  gave  1'451  gramme  of  neurokera- 
tin =  2902  per  cent. 
V.   50  grammes  of  the  pray  substance  (as  free  ns  possible  from  white) 
from  the  cortical  portion  of  the  cerebrum  of   the  same  man 
gave  0"1635  gramme  of  neurokeratin  =  0327  per  cent. 

The  gray  substance  was  separated  from  the  white  by 
scraping  instead  of  by  cutting,  a  method  which  enabled  us 
to  avoid  any  recognizable  quantity  of  white  matter. 


24  ON  NEUROKERATIN. 

In  this  connection  it  is  interesting  to  compare  the  re- 
sults of  determinations  in  which  a  portion  at  least  of  the 
myeline  bodies  were  removed  prior  to  digestion.  To  this 
end  the  material  (nerves  and  white  matter  from  the  cor- 
pus callosum)  was  first  placed  in  cold  alcohol,  the  nerves 
in  small  pieces,  while  the  brain  substance  was  rubbed  up 
with  the  alcohol.  This  alcohol  was  gradually  replaced  by 
fresh  quantities  of  absolute  alcohol,  with  which  the  tissue 
was  at  last  thoroughly  extracted  and  then  treated  with 
ether.  Much  of  the  fluid  could  be  poured  off  quite  clear, 
so  that  only  the  residue  remained  for  filtration,  for  which 
the  weighed  filter  was  at  "once  used.  After  the  removal  of 
the  ether  by  means  of  alcohol,  and  the  latter  by  cold  and 
finally  boiling  water,  the  substance  was  rinsed  from  the  fil- 
ter with  gastric  juice,  and,  after  being  rubbed  quite  fine, 
was  digested  with  the  fluid  in  a  beaker  for  fourteen  days. 
In  order  to  avoid  all  loss,  and  at  the  same  time  to  treat  all 
the  tissue  alike,  the  filter  in  its  funnel  was  filled  with  gas- 
tric juice,  the  end  of  the  tube  being  closed,  and  this  like- 
wise placed  in  the  digestion  oven.  During  the  digestion 
the  residue  was  several  times  ground  in  a  mortar,  precautions 
naturally  being  taken  to  prevent  loss.  When  the  digestion 
was  finished,  the  material  was  again  brought  upon  the 
filter  and  a  second  time  treated  as  already  desciibed  for  the 
complete  extraction  of  the  myeline  bodies.  Lastly,  the  nu- 
clein  was  removed  by  the  usual  method.  Following  are  the 
results  obtained : 

VI.  50  grammes  of  nerve  from  the  brachial  plexus  and  the  sciatic  nerve 

of  man  gave  O3005  gramme  of  neurokeratin  =  0-601  per  cent. 
VII.  50  grammes  of  white  substance  from  the  corpus  callosum  gave 
1-286  grammes  of  neurokeratin  =  2'572  per  cent. 

We  give  these  determinations  alone,  although  many 
others  were  made  more  especially  for  the  purpose  of  work- 


ON    NEUROKERATIN.  25 

in-- out  the  method.  As  is  Been  from  the  results,  the  sec- 
ond method  gives  for  the  nerves  (VI)  almost  doable  the 
amount  of  residue  fonnd  by  the  firsl  method  (I);  tor  the 

white  matter,  on  the  other  hand  (VII),  only  a  little  less 
residue  was  found  than  in  IV,  and  but  little  more  than  in 
III,  so  that  very  likely  in  a  larger  number  of  determina- 
tions little  difference  would  l>e  found.  There  is  no  ques- 
tion in  our  minds  as  to  the  cause  of  the  difference  in  the 
case  of  the  nerves;  it  is  in  great  part  dependent  upon  the 
fact  that  a  portion  of  the  connective  tissue  becomes  indi- 
gestible by  the  alcohol  treatment,  a  vie*  which  we  have 
proved  by  our  microscopic  study  of  the  question.  Another 
cause  for  the  difference  is  to  be  found  in  the  presence  of 
blood  in  the  nerves,  which  were  noticeably  red,  so  that  the 
neurokeratin  had  more  or  less  of  a  brownish  color,  and,  in 
fact,  contained  quite  a  little  iron.  In  the  white  substance 
of  the  corpus  callosum,  on  the  other  hand,  where  the  con- 
nective tissue  is,  without  question,  much  reduced  and  where 
no  blood  is  visible,  these  possible  admixtures  do  not  influ- 
ence the  amount  of  indigestible  residue. 

Further  confirmation  of  this  view  is  to  be  found  in  the 
determination  of  neurokeratin  made  by  Josephine  Cheva- 
lier in  1885,  a  piece  of  work  which  has  apparently  been 
wholly  ignored  by  histologists.  This  investigator  found  in 
the  fresh  sciatic  nerve  of  man  0*30  per  cent.*  of  neuro- 
keratin— a  result  which  shows  close  agreement  with  our 
finding  of  0316  per  cent.  Bearing  in  mind,  however,  that 
this  result  of  Chevalier's  was  obtained  from  nerve  fibers 
which  had  been  previously  freed  from  myeline  bodies,  it 
would  seem  at  first  glance  as  if  this  method  should  have 
yielded  double  the  amount  found,  in  correspondence  with 
our  determination  of  O'OOl  per  cent.  (Analysis  Yl).  The 
reason,  however,  is  to  be  found  in  the  fact  that  Chevalier, 

*  Zeitschrift  fur  physiol,  Clieni.,  x,  p.  100. 
i 


26  ON  NEUROKERATIN. 

before  digesting  the  myeline  free  tissue  in  gastric  juice,  re- 
moved the  connective  tissue  which  had  become  indigestible 
through  the  alcohol-ether  treatment  by  heating  it  with 
water  at  120°  C.  in  a  sealed  tube  for  twelve  hours. 

The  distribution  of  neurokeratin  in  the  brain  is  quite 
remarkable.  In  no  portion  of  the  mammalian  brain  is  there 
gray  substance  to  be  found  which  does  not  contain  medul- 
lated  nerve  fibers ;  but  in  the  gray  cortex  of  the  cerebrum 
the  admixture  is  quite  limited,  and  in  the  corpus  collosum 
white  nerve  fibers  are  present,  with  extremely  well  devel- 
oped and  thick,  spongy  structure,  and  but  little  true  neurog- 
lia. The  cortex  of  the  cerebellum  (compare  Analysis  II), 
on  the  other  hand,  is  composed  of  gray  matter  in  which 
many  medullated  nerve  fibers  are  to  be  found.  The  small 
amount  of  neurokeratin,  only  0*312  per  cent. — somewhat 
smaller  even  than  that  found  in  the  nerves — would  well 
agree  with  this  view.  In  the  cortex  of  the  cerebrum  the 
content  of  neurokeratin  is  somewhat  higher  than  in  nerves — 
viz.,  about  0-011  per  cent. — too  high,  probably,  to  be  ex- 
plained on  the  ground  of  admixture  of  medullated  nerve 
fibers.  Thin  sections  of  the  cortex,  freed  from  myeline 
substance,  prepared  and  treated  according  to  a  method  to 
be  described  later  in  connection  with  nerves,  have  shown 
us  beyond  question  the  presence  of  a  fine,  spongy  network 
of  horny  matter,  developed  thickest  directly  under  the  pia. 

The  richness  of  the  white  substance  in  neurokeratin  is 
quite  remarkable,  the  amount  being  about  nine  times  that 
contained  in  the  peripheral  nerves.  Hence  the  brain  is 
naturally  the  best  material  from  which,  to  prepare  large 
quantities  of  this  substance.  The  great  shrinkage  of  the 
connective  tissue  and  the  presence  of  the  sponge-like  net- 
work of  horny  matter  all  through  the  medulla  find  their 
chemical  expression  in  the  great  preponderance  of  neuro- 
keratin. 


ON    NEUROKERATIN.  27 

In  order  to  comprehend  fully  the  significance  of  the 
above  percentages,  they  need  to  be  considered  in  connec- 
tion with  the  amount  of  water  and  of  the  so-called  myeline 

substances  of  the  tissue.  According  to  <  'he\  alter,  the  ner\  eta 
of  man  contain  3372  per  cent,  of  dry  Babstance,  of  which 
10-55  parts  are  insoluble  in  alcohol  and  ether.  Thia  mye- 
line-free substance  would  therefore,  according  to  Analysis 
I,  contain  1-909  per  cent,  of  neurokeratin.  The  amount  of 
neurokeratin  contained  in  the  myeline-free  nerve  fibers, 
however,  would  be  somewhat  higher  than  tlii-,  as  the  dry 
substance  necessarily  contains  some  accessory  tissues.  Che- 
valier places  it  at  3*07  per  cent.  For  the  human  brain  it  is 
somewhat  hazardous  to  estimate  the  content  of  water,  since 
the  well-known  analyses  of  the  gray  cortex  by  v.  Bibra, 
Birkner,  Bourgoin,  and  Weissbach  show  differences  of  82*25 
to  84*97  per  cent.,  and  in  the  case  of  the  white  substance 
of  63*54  to  73*93  per  cent,  of  water.  The  average  of  all  the 
analyses  would  indicate  a  content  for  the  gray  substance  of 
84  per  cent,  in  round  numbers,  and  of  70  per  cent,  for  the 
white  substance.  Determinations  of  the  myeline  bodies  in 
both  the  gray  and  white  matter  have  been  made  only  by 
Petrowski,*  and  the  material  used  in  his  experiments  was 
ox  brains.  Converting  his  results,  expressed  in  percentages 
of  dry  substance  to  the  moist  weight  of  the  human  brain, 
we  find  for  the  gray  matter  10*15  per  cent,  of  myeline-free 
substance,  and  for  the  white  matter  8*59  per  cent.  On  the 
basis  of  Analysis  V,  the  former  would  therefore  contain 
3-22  per  cent,  of  neurokeratin,  while  the  white  matter,  ac- 
cording to  Analysis  TV,  would  contain  the  enormous  quan- 
tity of  33*77  per  cent,  of  neurokeratin.  Of  the  cerebellum 
we  have  not  been  able  to  find  any  analysis  whatever,  but, 
assuming  that  its  content  of  water  and  myeline  bodies  is 
the  same  as  in  the  cortex  of  the  cerebrum,  then   its  mve- 

*  Arch.  f.  d.  ges.  Physiol.,  vii,  p.  367. 


28  ON  NEUROKERATIN. 

line-free  cortex  would  contain  (Analysis  II)  3*07  per  cent, 
of  neurokeratin. 

Hence  our  results  indicate  : 

For  rnyeline-free  dry  nerve  substance.. . .     1*91  per  cent,  neurokeratin. 

For  myeline-free  dry  gray  substance ... .     3*22    "      " 

For  myeline-free  dry  white  substance  .  . .   33-77    "      "  " 

The  old  estimate  that  neurokeratin  was  present  in  the 
whole  brain  to  the  extent  of  at  least  15  to  20  per  cent,  of 
the  myeline-free  dry  substance  was,  therefore,  none  too 
high. 

In  order  to  assure  ourselves  that  in  these  determinations 
there  was  no  great  error  from  the  peculiarities  of  the  meth- 
od, especially  as  at  times  the  weight  of  the  filter  was  quite 
large  in  proportion  to  the  amount  of  the  undigested  residue 
(0*9  to  2-7  grammes),  we  have  applied  the  method  to  sev- 
eral other  organs,  using  for  this  purpose  the  kidneys  and 
liver. 

Forty-five  grammes  of  fresh  liver,  with  the  usual  amount 
of  blood,  from  a  recently  killed  rabbit,  was  first  digested 
with  gastric  and  pancreatic  juice,  then  treated  with  alcohol, 
ether,  and  one-per-cent.  caustic  soda.  The  final  amount  of 
residue  obtained  was  0-0263  gramme  ==  0*058  per  cent.,  or  a 
little  less  than  one  fifth  the  smallest  amount  of  neurokeratin 
found  in  the  cortex  of  the  cerebellum. 

Ten  grammes  of  dry  substance  from  a  calf's  liver,  almost 
free  from  blood,  was  rubbed  up  with  alcohol,  then  extracted 
with  ether,  etc.,  and,  after  thorough  digestion  and  washing 
with  boiling  water,  left  a  residue  so  small  as  to  be  un- 
weighable. 

Thirteen  grammes  of  fresh  kidney  from  a  rabbit,  on  being 
treated  in  the  same  manner,  left  a  residue  of  0*0092  gramme 
=  0*070  per  cent. — somewhat  larger  than  the  residue  left  by 
the  fresh  liver,  but  less  than  one  fourth  the  residue  from 


ON   NEUROKERATIN.  29 

the  cerebellum.  The  residue  had  a  grayish  look  and  be- 
came blackish  brown  on  boiling  with  acetate  of  lead  and 

caustic  soda.  As  the  kidneys  were  omitted  in  an  earlier 
investigation  *  of  the  indigestible  constituents  of  various 
organs,  a  further  examination  of  this  small  residue  would  be 
desirable. 

Microscopic  Detection  of  Neurokeratin  in  Nerve  Fibers. 
— The  following  description  is  intended  to  serve  princi- 
pally as  a  guide  to  the  detection  of  this  peculiar  insoluble 
portion  of  the  nerve  fiber  which  remains  after  all  else  has 
been  dissolved  away,  rather  than  as  a  statement  of  the 
natural  form  and  arrangement  of  neurokeratin  in  the  living 
fiber. 

Neurokeratin  was  first  described  as  having  the  form  of 
a  double  sheath,  of  which  the  outer  one  surrounds  the 
medulla  of  the  nerve  under  the  sheath  of  Schwann,  while 
the  inner  incloses  the  axis  cylinder,  both  being  joined  to- 
gether by  means  of  a  peculiar  knotty  structure.  It  was  not 
assumed  that  the  knotty  forms  corresponded  exactly  with 
those  pre-existent  in  life,  but  it  was  plainly  stated  f  that 
the  prolongations  of  the  outer  horny  sheath  extend  to  the 
iuner,  under  certain  circumstances,  in  the  shape  of  the  well- 
known  spindle  aud  funnel  forms  of  the  medullary  sheath. 
When  nerves  are  treated  with  alcohol,  from  the  condition 
of  things  normally  present  in  the  medullary  space,  their  ap- 
pearance counts  for  little,  since  the  naturally  thick  axis 
cylinder  of  the  fiber  shrivels  and  the  small  medullary  mantle 
or  sheath  becomes  thicker,  through  the  action  of  the  alcohol. 
Further,  bridge-like  connections  must  extend  between  these 
two  horny  sheaths,  and  these  might  thereby  be  broken.  Ob- 
viously, therefore,  for  the  first  step  in  the  treatment  of  the 

*  VerhandL  d.  oaturhist.  med.  Vereins  zu  Heidelberg,  X.  F.,  Band  I, 
pp.  451-456. 

f  Jbid.,  p.  461. 


30  ON   NEUROKERATIN. 

fiber,  only  those  reagents  can  be  employed  which  will  retain 
the  natural  volume  of  the  axis-cylinder — as,  for  example, 
osmic  acid,  which,  however,  we  have  not  as  yet  used  in  con- 
junction with  the  treatment  by  digestion  and  extraction  of 
the  myeline  bodies.  Suggestions  have  indeed  been  made  as 
to  the  actual  way  in  which  the  keratin  network  is  formed 
and  the  manner  of  its  arrangement,  but  they  relate  to  micro- 
scopical forms  from  which  neither  the  myeline  bodies  nor  the 
digestible  matter  of  the  nerve  were  completely  extracted. 
We  refer  to  the  so-called  neurokeratin  funnel  and  spiral 
forms  described  by  several  Italian  observers,  and  to  the  well- 
known  appearance  of  transverse  sections  of  the  medulla  in 
osmic-acid  preparations  of  nerves.  These,  recently  ob- 
served by  Joseph,  but  not  wholly  comprehended  in  their 
complexity,  were  long  known  to  us  as  beautiful,  untinted 
figures,  which  make  their  appearance  in  transverse  sections 
of  mammalian  nerves,  within  the  stained  (by  osmic  acid) 
mantle  of  the  medulla. 

The  preparations  were  most  advantageously  obtained 
by  extraction  of  the  myeline  substance  and  digestion  of  the 
teased  nerve  on  a  glass  slide.  For  this  purpose  it  is  of 
some  importance  what  kind  of  animal  the  nerves  are  taken 
from.  In  the  nerves  of  the  frog,  and  in  those  from  fishes 
likewise,  certain  differences  are  to  be  observed,  so  that 
the  following  description,  except  when  otherwise  specially 
stated,  refers  solely  to  mammalian  nerves,  and  particularly 
to  those  from  rabbits,  which  appear  best  adapted  for  the 
purpose. 

As  in  the  preparation  of  neurokeratin  in  quantity,  so  in 
the  microscopical  preparation  there  are  two  methods  of 
treatment,  in  one  of  which  the  extraction  of  the  myeline 
substance  precedes  the  digestion,  while  in  the  other  the  latter 
process  is  applied  first.  We  think  it  best,  however,  to  de- 
scribe somewhat  in  detail  both  processes,  since  histologists 


ON   NEUBOKEB \n\  31 

appear  to  be  divided  into  two  groups  concerning  tliis  ques- 
tion,  the  one  maintaining  that  undigestiblc  structnrea  are 
to  be  found  in  nerve  fibers  only  when  the  myeline  bodies 
have  been  removed  by  alcohol  prior  to  digestion,  and  thai 
when  the  order  of  treatment  is  reversed  Bach  structures  are 
not  to  be  seen;  the  other  group,  on  the  contrary,  contending 
that  it  is  impossible  to  find  Buch  andigestible  Btrnctnres  in 
nerve  fibers  under  any  circumstances  whatever. 

We  will  firsl  call  attention  to  the  methods  of  treatment 
common  to  both  processes,  and  to  some  of  the  precautions 

necessary  to  lie  taken. 

1.  The  complete  extraction  of  the  myeline  substance,  the 
difficulties  of  which  have  already  been  dwelt  upon,  is  not 
easily  accomplished,  even  in  microscopical  preparations 
where  longitudinal  and  transverse  sections  of  the  nerve  are 
only  15  to  20  /*,  thick.  We  have  indeed  found  it  so  diffi- 
cult that  we  hnve  heen  impressed  by  the  lack  of  any  state- 
ment to  this  effect  in  the  many  writings  on  tliis  and  kindred 
subjects,  in  which  this  circumstance  must  be  of  consider- 
able importance.  These  residues  of  myeline  bodies  are 
found  in  the  nerve  fibers,  either  in  the  form  of  glistening, 
oval-shaped  balls — which,  from  their  size  and  distribution, 
might  be  taken  for  nuclei — or  in  the  form  of  a  symmetrical 
thickening  of  the  keratin  network,  comparable  to  a  layer  of 
varnish,  thus  imparting  to  the  structure  more  luster  and 
rendering  it  more  refractive.  This  residue  of  myeline  mat- 
ter is  in  all  cases  composed  of  cerebrin,  which  is  n<>t  easily 
soluble  even  in  boiling  alcohol  or  benzol,  but,  on  the  con- 
trary, dissolves  out  slowly,  quickly  separating  from  the  fluid 
as  it  cools.  It  is  necessary,  therefore,  after  the  preliminary 
treatment  of  the  preparation  with  cold  alcohol  and  ether,  to 
bod  it  with  both  alcohol  and  benzol  for  five  to  ten  minutes, 
at  least  three  times,  and  to  pour  off  the  fluid  each  time 
while  it  is  still  hot,  for  the  cerebrin  shows  a  great  tendency 


32  ON  NEUROKERATIN. 

to  separate  and  adhere  to  the  particles  of  tissue,  even  when 
the  fluid  itself  shows  no  sign  of  turbidity. 

2.  The  digestion  of  the  nerve  fiber  we  have  in  some  cases 
carried  on  in  large  glass  dishes,  while  at  other  times  we 
have  used  small  test  tubes  35  to  45  mm.  long  and  15  to  20 
mm.  wide,  loosely  corked  and  set  in  a  metal  framework  in 
a  warm  chamber,  the  temperature  of  which  ranged  from 
37°  to  41°  C.  The  tubes  with  their  contents  of  5  to  10  c.  c. 
were  shaken  as  often  as  practicable,  taking  care,  however, 
that  the  sections  or  small  pieces  of  nerve  are  not  broken  by 
too  vigorous  agitation.  In  fact,  there  should  be  a  rotation 
of  the  contents  of  the  tube  rather  than  a  shaking,  thus  pre- 
venting at  the  same  time  the  preparations  from  coming  in 
contact  with  the  cork,  to  which  they  are  inclined  to  adhere. 
As  to  the  length  of  time  necessary  to  continue  the  digestion, 
probably  twenty  four  hours  in  nearly  all  cases  would  be  suf- 
ficient; we  have,  however,  generally  continued  the  process 
much  beyond  this  length  of  time,  usually  for  a  week,  and 
in  some  cases  have  even  extended  the  time  to  seven  weeks. 
More  important,  however,  than  the  element  of  time  is  the 
condition  of  the  digestive  fluid  and  the  frequency  with 
which  it  is  changed.  Five  different  kinds  of  gastric  juice, 
representing  as  many  different  methods  of  preparation,  were 
used  on  the  same  object,  one  after  the  other:  1,  two  gly- 
cerin mixtures,  the  first  composed  of  -|-,  the  second  of  -| 
pepsin-glycerin  (prepared  by  us  and  known  to  be  of  maxi- 
mum activity) ;  2,  a  hydrochloric-acid  extract  of  the  mucous 
membrane  of  a  pig's  stomach,  prepared  by  self-digestion  at 
40°  C ;  3,  same  as  2,  only  prepared  in  the  cold,  both  with 
-X  mucous  membrane  ;  4,  same  as  2,  with  fa  mucous  mem- 
brane. All  of  the  mixtures  contained  0*4  per  cent.  HO. 
Each  of  these  digestive  fluids,  when  warm,  dissolved  raw  fibrin 
almost  instantaneously  even  when  they  were  a  month  or  more 
old.     According  to  our  experience,  artificial  gastric  juice 


ON    NEUROKERATIN.  33 

prepared  like  the  above  and  preserved  in  loosely  stoppered 
flasks  in  a  cool  place,  with  a  trace  of  thymol  added,  remains 
good  for  an  exceedingly  long  time  without  loss  of  digestive 
power.  All  of  our  experiments,  however,  have  been  du- 
plicated with  non-thymolized  gastric  juice  preserved  in 
the  cold  air  of  winter,  the  digestions  themselves  being  car- 
ried on  in  a  warm  chamber,  the  air  of  which  was  kept 
strongly  permeated  with  thymol.  The  trypsin  solution  was 
the  same  in  all  experiments;  it  always  contained  0'5-per- 
cent.  sodium  carbonate,  was  repeatedly  filtered  from  the 
separated  ty rosin,  saturated  with  thymol  at  the  temperature 
of  the  room,  and  digested  raw  fibrin  very  noticeably  in  five 
minutes,  completely  in  ten  minutes.  It  was  not  so  power- 
ful in  digestive  action  as  it  is  possible  to  prepare  such  a 
fluid,  but  was  far  more  active  than  was  necessary  for  the 
purpose  in  view.  At  the  end  of  each  digestion  a  portion  of 
the  fluid  was  always  tested  with  fibrin  in  order  to  be  sure 
that  its  energy  was  unimpaired. 

When  the  digestion  of  the  object  is  completed  it  be- 
comes in  many  cases  a  matter  of  some  importance,  owing  to 
the  small  size  of  the  preparation  and  its  great  tenderness,  to 
transfer  it  properly  to  the  glass  slide  for  examination.  This 
we  have  most  successfully  accomplished  by  placing  the  small 
test  tube  containing  the  object  in  a  trough,  filled  with  water, 
made  of  glass  plates  fused  together,  whereby  the  fine  sedi- 
ment is  made  more  plainly  visible  on  the  curved  bottom 
of  the  glass,  whence  it  can  be  fished  out  with  a  suitable 
pipette.  The  same  method  can  be  advantageously  used 
when  it  is  necessary  to  extract  the  preparation  with  caustic 
soda  or  other  reagents,  or  to  wash  it  with  water.  The  ex- 
traction of  the  preparation  with  caustic  alkali  for  the  re- 
moval of  nuclein  we  have  invariably  performed,  although  it 
does  not  alter  the  microscopical  appearance  of  the  object, 
since  the   nuclein    in   these   cases   comes   entirely  from   the 


34  ON   NEUROKERATIN. 

residues  of  the  nuclei.  As  a  preserving  medium  we  have 
employed  very  dilute  glycerin,  first  staining  the  more  ten- 
der objects  with  Delafield's  hematoxylin. 

I.  Nerves  digested  after  Extraction  of  the  Myeline  Sub- 
stance.— The  nerves  are  first  stretched  out  by  means  of 
threads  in  small  glass  tubes,  or  else  fastened  upon  corks  and 
then  hardened  in  cold  alcohol.  After  twenty-four  hours 
they  are  cut  into  small  pieces  from  1  to  3  cm.  long,  and 
then  freed  from  myeline  matter,  as  already  explained,  by 
boiling  with  alcohol  and  benzol  and  also  with  ether.  From 
a  mixture  of  alcohol  and  ether  the  pieces  are  transferred  to 
a  solution  of  celloidin  of  gradually  increased  strength.  After 
the  hardening  of  the  celloidin  in  seventy-per-cent.  alcohol 
the  pieces  are  cut  by  means  of  the  microtome  into  longi- 
tudinal and  transverse  sections  15  to  20  /x,  thick,  which  are 
then  further  extracted  with  the  above  reagents.  While  in- 
closed in  the  celloidin  lamellae  the  tissue  can  not  be  treated 
with  digestive  fluids  to  advantage,  since  the  smallest  quan- 
tity of  this  substance  renders  both  gastric  and  pancreatic 
juice  wholly  inactive,  which  fact  is  to  be  attributed  to  the 
peculiarity  possessed  by  nitrocellulose,  discovered  by  Dani- 
lewsky,*  of  precipitating  the  digestive  enzymes.  We  found 
that  a  few  thin  shavings  of  celloidin,  washed  simply  with  wa- 
ter and  placed  even  in  large  quantities  of  the  digestive  fluid, 
freed  it  so  completely  from  the  enzyme  that  its  digestive 
power  was  almost  wholly  impaired.  In  gastric  juice  it 
would  appear  at  the  first  glance  as  if  transverse  sections  of 
a  nerve  do  really  dissolve,  for  after  a  time  it  will  be  noticed 
that  a  fine  hole  appears  in  the  celloidin  lamella,  where  the 
nerve  section  is  known  to  be.  On  close  scrutiny,  however, 
it  will  be  found  that  the  entire  tissue  still  remains,  but  ren- 
dered more  or  less  transparent  on  the  edge  by  the  swelling 
of  the  connective  tissue  in  the  acid.     That  the  gastric  juice 

*  Virchow's  Archiv,  xxxv,  p.  2*79. 


ON   NEUROKERATIN.  35 

has  really  lost  all  of  its  digestive  power  can  he  easiU  >\\<>w  n 
by  placing  in  it  a  piece  of  fibrin,  which  will  he  seen  to  swell 
up  and  hecoinc  more  or  less  transparent  without,  however, 
dissolving.  The  nerve  sections  must  therefore  be  entirely 
freed  from  celloidin,  and  this  operation  serves  likewise  to 
remove  all  remaining  traces  of  myeline  matter.  For  this 
purpose  the  sections  are  treated  for  twenty-four  hours  with 
alcohol  and  ether  and  then  again  extracted  with  hot  alcohol, 
benzol,  etc.,  as  previously  descrihed,  after  which  they  are 
freed  from  alcohol  by  washing  with  water  and  then  subjected 
to  digestion. 

The  digestion  can  be  carried  out  in  four  different  ways  : 
1,  with  gastric  juice  ;  2,  with  pancreatic  juice  ;  3,  with  pan- 
creatic juice  after  the  sections  have  been  previously  boiled 
with  water ;  4,  with  pancreatic  juice  after  previous  treat- 
ment with  gastric  juice  or  dilute  hydrochloric  acid.  The 
third  and  fourth  methods  give  the  same  results  as  the  first, 
since  from  the  preliminary  treatment  the  connective  tissue 
is  rendered  wholly  soluble  in  the  trypsin  solution.  The  sec- 
ond method,  on  the  contrary,  preserves  the  collagenous  fibers 
entire,  together  with  the  neurokeratin.  An  exception,  how- 
ever, is  to  be  noticed  in  this  connection  in  the  nerves  of  the 
frog.  As  Aug.  Ewald  *  has  recently  observed,  the  tendons 
of  the  frog,  in  distinction  from  those  of  the  Mam  ma!  in,  are 
decidedly  altered  by  trypsin,  and  the  same  is  true,  though 
to  a  less  extent,  of  the  connective  tissue  contained  in  the 
nerves  of  the  frog.  After  a  long  digestion  with  trypsin  the 
nerve  fibers  are  seen  to  fall  almost  entirely  apart,  and  it  ap- 
parently makes  no  difference  whether  the  nerve  is  fresh  or 
has  been  previously  extracted  with  alcohol.  In  both  cases 
the  nerve,  on  treatment  with  warm  trypsin  solutions,  softens 
and  the  fibers  separate,  while  the  collagenous  fibers  which 
bound  them  together,  and  which  at  first  can   be  readily  iso- 

*  Zeitschrift  fur  Biologic,  xxvi,  p.  1. 


36  ON   NEUROKERATIN. 

lated,  after  a  time  disappear,  and  in  their  place  is  to  be  found 
some  soft,  slimy  matter  or  a  membranous  mass,  the  latter 
coming  from  the  thicker  neurilemma.  The  usual  differ- 
ence, therefore,  in  the  character  of  the  residue  left  by  agas- 
tric and  pancreatic  digestion  disappears  in  the  case  of  the 
frog's  nerves,  a  fact  which  has  been  of  some  advantage  to 
us  in  our  study  of  this  matter,  as  will  presently  be  seen. 

In  the  mammalia  this  order  of  things  is  in  a  certain  sense 
reversed,  for  here  the  connective  tissue  is  so  altered  by  the 
agents  employed  for  the  extraction  of  the  myeline  substances 
that  it  is  not  completely  broken  up  by  gastric  juice,  even 
after  previous  boiling  with  water,  or  by  trypsin  digestion 
when  treated  as  described  in  3  and  4.  Herein  lies  the 
probable  cause  of  the  different  results  obtained  in  the 
quantitative  determination  of  neurokeratin,  both  in  nerves 
and  in  brain  tissue,  when  the  extraction  of  the  myeline 
bodies  precedes  the  digestion  process  instead  of  following 
it.  Furthermore,  this  explains  why  microscopical  prep- 
arations of  neurokeratin  are  more  readily  obtained  and  in 
better  condition  from  nerves  which  have  been  previously 
extracted  with  alcohol,  etc.,  prior  to  digestion,  since  under 
such  circumstances  there  remains  after  the  latter  process 
something  which  holds  together  the  residue  of  the  fibers. 

At  the  same  time  such  preparations  of  nerve  fibers  are 
seen  to  be  free  from  any  accompanying  tissue  of  recogniza- 
ble structure ;  that  which  lies  between  the  fibers  is  hardly 
recognizable  as  a  cementing  substance,  for  it  is  only  on  the 
edges  of  the  section  that  the  almost  homogeneous  border 
of  transparent  matter  can  be  detected,  showing  here  much 
like  an  outer  sheath,  thick  and  transparent.  Another  con- 
stituent often  noticed  is  broken  residues  of  the  red  blood- 
corpuscles,  which  have  been  rendered  indigestible  by  treat- 
ment with  alcohol.  They  often  occur  in  rows,  corresponding 
to  the  digested. blood- vessels,  in  between  the  residues  of  the 


OS    NEUROKERATIN.  37 

nerve  fibers.  Of  the  nerve  fibers  themselves,  many  are  com- 
pletely dissolved,  aside  fr<>m  the  residues  already  mentioned, 
and  these  especially  show  what  the  arrangement  <>f  the 
neurokeratin  in  the  Hher  is.  They  are  extremely  friable, 
and  are  reduced  to  cylindrical,  wrinkled  Btrings  or  cords,  in 
which  can  frequently  be  seen  an  inner  string,  like  a  wick  in 
a  candle,  while  surrounding  the  latter  is  a  rumpled  mass 
uniting  it  with  the  outer  covering.  We  have  treated  hun- 
dreds of  Bnch  sections,  and  in  many  cases  have  allowed 
them  to  lie  for  months  in  a  large  excess  of  thymolized  <_r,i— 
trie  juice  in  small  test  tubes,  and  in  no  case  have  we  tailed 
to  find  the  neurokeratin  residue,  and  this  both  in  large 
and  small  nerves  alike.  Further,  in  every  case  we  have 
proved  the  great  strength  of  the  gastric  juice  emploved  by 
testing  its  action  on  fibrin  which  had  been  boiled  in  water 
and  also  treated  with  alcohol  and  ether. 

Preparations  which  have  been  treated  with  trypsin,  after 
the  manner  given  under  2,  are  less  changed  than  the  pre- 
ceding, and  retain  the  natural  form  of  the  neurokeratin 
better,  since  in  these  all  of  the  collagenous  fibers,  which 
hold  the  nerve  fibers  together,  remain,  and,  furthermore,  are 
not  swollen  as  when  treated  with  the  acid  gastric  juice.  In 
addition,  the  nerve  fibers  themselves  are  less  deformed  than 
when  treated  with  acid  fluids,  since  the  swelling  action  of 
the  acid  on  the  connective  tissue  fibers  tends  to  shorten 
longitudinal  sections  of  nerves,  and  hence  twists  and  crum- 
ples the  nerve  fibers  themselves.  Generally  the  nerve 
fibers  lie  in  straight  rows,  showing  for  long  stretches  an 
outer  shell,  together  with  an  inner  string  of  the  shrunken 
horny  sheath  of  the  axis  cylinder  and  the  connecting  struct- 
ure. In  many  places,  however,  there  may  be  found  por- 
tions of  fibers  in  which  it  is  impossible  to  r< gnizc  the 

individual  parts  of  the  horny  sheaths,  where  the  fiber  re- 
sembles a  chain  of  crumpled  fragments,  owing  to  the  coin- 


38  ON  NEUROKERATIN. 

plete  solution  of  the  contents  of  the  sheaths  and  the  elastic 
contraction  of  the  neurokeratin,  for,  when  all  of  the  sub- 
stance naturally  filling  up  the  space  of  the  fiber  is  removed, 
no  resistance  is  offered  to  the  elasticity  of  the  neurokeratin, 
and  hence  it  falls  together  more  or  less  as  one  mass. 

If  mammalian  nerves  are  boiled  with  water  or  treated 
with  acid,  previous  to  subjecting  them  to  the  action  of 
trypsin  after  the  method  under  3  and  4,  they  show  the 
same  appearance  as  when  digested  in  gastric  juice.  More- 
over, fibers  which  have  been  digested  in  gastric  juice  are 
not  apparently  further  changed  by  after-treatment  with 
pancreatic  juice.  There  is,  therefore,  a  certain  residue  or 
decomposition  product  of  the  mammalian  connective  tissue 
present  in  the  nerves  after  extraction  of  the  myeline  sub- 
stances, which  resists  the  action  of  both  digestive  fer- 
ments. 

II.  Nerves  digested  prior  to  Extraction  of  the  Myeline 
Substances. — The  character  of  the  residue  remaining  after 
this  method  of  treatment  has  long  been  known,  and  from 
the  very  beginning  much  weight  has  been  attached  to  its 
peculiarities.  The  process,  however,  like  that  employed  in 
the  preceding  case,  is  not  so  easy  but  that  considerable 
practice  is  necessary  to  avoid  loss  of  the  undigestible 
residue  by  its  floating  off  from  the  glass  slide,  or  to  obtain 
good  preparations  from  the  brittle  mass  left  at  the  comple- 
tion of  the  process.  Moreover,  the  conditions  are  not  as 
favorable  here  for  obtaining  the  horny  matter  in  its  natural 
form  as  where  the  digestion  follows  the  alcohol  treatment, 
for  in  the  latter  process  the  fluid  portion  of  the  medulla, 
especially  that  containing  the  albuminous  matter,  is  first 
coagulated  and  hardened.  Then,  when  the  myeline  bodies 
are  removed,  there  remains  a  horny  mass  covered  over  or 
penetrated  by  the  coagulated  albumin  and  showing  a  form 
identical  with  the  compact  structure  long  ago  described  by 


ox  NEUROKERATIN.  39 

Henle  and  Merkcl,*  whicli  many  later  observers  have  held 
as  congruent  with  neurokeratin,  a  mistake  which  Ilumpf 
has  already  called  attention  to.  However,  this  preliminary 
coagulation  of  the  proteid  matter  has  an  advantage  for  the 
digestive  process  in  that  it  helps  to  fix  the  neurokeratin, 
and,  further,  the  coagulated  albumin  is  only  slowly  softened 
by  the  digestive  juice,  so  that  for  this  reason  it  ie  possible 
to  obtain  the  microscopic  forms  in  much  better  condition 
than  in  the  other  method.  In  the  direct  digestion  of  fresh 
nerves,  on  the  other  hand,  only  soft  matter  fills  out  and  sur- 
rounds the  neurokeratin,  from  which  the  latter  can  find 
little  or  no  support,  so  that  in  the  rapid  digestion  and  solu- 
tion of  the  non-coagulated  albumin  the  horny  residues  natu- 
rally fall  together  and  appear  extremely  rumpled  and 
twisted.  On  being  digested  with  gastric  juice,  a  very 
marked  shortening  and  swelling  occur  in  fresh  nerves,  the 
extent  of  which  can  be  estimated  from  the  corresponding 
overflow  of  the  medulla  from  the  mushroom-like  end  of 
each  piece  of  nerve.  When  all  of  the  albuminous  matter 
has  been  rendered  non-coagulable  by  digestion  and  the 
inyeline  bodies  extracted  from  the  nerve,  it  must  not  be 
expected  that  the  residue  will  show  more  than  a  confused 
appearance,  for  it  is  only  by  the  greatest  care  in  the  hand- 
ling of  the  object  under  the  cover-glass  that  it  will  be 
possible  in  some  measure  to  arrange  the  parts  for  observa- 
tion. 

If  fresh  nerves  are  digested  with  gastric  juice  in  small 
test  tubes  and  the  medullary  substances  then  removed  by 
the  ordinary  method,  the  residue  will  be  decidedly  broken 
and  mixed,  for  when  the  cerebrin  is  dissolved  out  by  boil- 
ing with  alcohol  and  benzol,  the  pieces  become  by  this  treat- 
ment so  brittle  that  on   the   boiling  of  the   fluid    they  are 

*  Zeitschr.  f.  rat.  Med.,  xxxiv,  p.  49,  Table  V,  RgB.  20  and  21. 


40  ON"   NEUROKERATIN. 

broken  apart — so  much  so  indeed  that  they  can  not  be  taken 
hold  of  by  forceps  or  needles,  but  can  be  caught  only  by 
decantation  of  the  fluid  and  the  use  of  wide  pipettes.  The 
residue  thus  obtained,  however,  always  possesses  the  same 
characteristic  properties,  as  any  one  can  see  who  will  take 
the  necessary  pains  in  manipulation — viz.,  insolubility  in 
caustic  soda,  intense  coloration  with  hematoxylin,  non- 
digestibility  in  gastric  and  pancreatic  juice,  etc. 

A  far  better  method  of  obtaining  neurokeratin  for  mi- 
croscopic study,  and  one  which  yields  the  product  in  much 
better  condition,  consists  in  the  following  treatment :  A 
piece  of  the  sciatic  nerve  from  a  rabbit,  2  to  3  cm.  long, 
and  which  has  been  digested  for  one  to  seven  weeks  in  100 
c.  c.  of  active  gastric  juice,  is  thoroughly  washed  with  water, 
then  placed  in  alcohol  of  gradually  increased  strength,  then 
in  ether,  in  alcohol-ether,  and  finally  in  celloidin  of  gradu- 
ally increased  concentration.  In  making  these  transfers 
from  one  fluid  to  another  it  is  not  necessary  to  use  a  pipette, 
which  might  possibly  crush  the  brittle  residue  of  the  nerve, 
but  it  is  better  accomplished  by  careful  pouring  of  the 
fluids,  and,  when  necessary,  by  cautious  slipping  of  the  bit 
of  tissue.  The  piece  of  nerve  is  then  hardened  with  the 
celloidin  in  70-per-cent.  alcohol  and  cut  into  sections  with 
the  microtome.  Longitudinal  sections  of  the  nerve  3  to  5 
mm.  long  are  best  adapted  for  study.  The  celloidin  sec- 
tions are  taken  from  the  70-per-cent.  alcohol  singly  by 
means  of  pinchers  and  placed  in  absolute  alcohol,  where  they 
are  allowed  to  remain  until  they  begin  to  soften  on  the 
edges.  This  may  be  considered  as  evidence  that  they  are 
sufficiently  dehydrated  to  be  boiled  in  benzol,  by  means  of 
which  the  remainder  of  the  myeline  bodies,  especially  the 
cerebrin,  is  completely  dissolved  without  alteration  of  the 
celloidin.  From  the  hot  benzol  the  celloidin  sections  are 
placed  in  cold  benzol,  then  again  in  absolute  alcohol  until 


BOKBRATDI  41 

they  show  a  taadei  (ten,  then  in  dilute  :.naJ- 

ly  in  water  and  in  gljeerin,  in  the  latter  of  which  the  \ 
aration  can  be  best  examined.     In  this  manner  all 
and  rnmhrag  are   avoided  and  a  preparaJ  -  .tied 

which,  while  in  great  part  composed  of  twisted  and  k; 

-     ?,  still  s         -  many  pieces  which  may  be  ea-ily  r. 
nized   as   residues  >>t"  ner\e   fibers,   with   the   char 
joining  of  the  outer  and  inner  horny  sheaths  and  ti 
rk  of  neurokeratin. 

In  order  te  exclude  all  doul       B       "he  eompleten  — 
the  extraction  of  the  myeliue  bodies,  the  celloidin.  which 
still  surrouuds  the  residue  from  the  nerve,  can  be  complete- 
ly removed.    This  is  best  accomplished,  after  the  final  wash- 
_   with  alcohol,  by  dropping  upon  the  softened  section 
placed  on  a  h.  '  _   tss  slide  boiling  alcohol,  and  then 
the  residue  with  ether,  care  being  taken  to  avoid  its  :; 
ing  away.     Addition  of   1   to-5-per-cent.  car.-        -         will 
then  show  the  character  of  the  insoluble  substauee.     The 
best  preparations  are  obviously  obtained  from  regular  1.  I  _ 
tudinal  sections  of  nerves,  while  trausverse  and  oblique  -     - 
lions  are  more  apt  to  yield  poorer  and  less  satisfactory  speci- 
mens. 

For  mieroscopists  we  would  on  some  accounts  recom- 
mend the  pancreatic  instead  of  gastric  digestion,  in  which 
the  collagenous  fibers  take  the  place  of  celloidin  in  prevent- 
ing the  eutire  collapse  of  the  nerve  fiber  in  the  after-treat- 
ment with  alcohol,  benzol,  etc..  and  in  which,  likewise,  the 
volume  and  length  of  the  fiber  remain  practically  unaltered. 
Furthermore,  mammalian  nerves  can  remain  in  the  warm 
trypsin  solution  as  long  aa  desired,  and  that  too  without 
g  ition  of  the  mixture.  The  pieces  can  then  be  washed 
iu  water,  freed  from  myelitic  bodies,  spread  upon  appropri- 
I  the  fibers  teased  apart  in  the  ordinary  tray. 
One  would  hardly  expect  to  rind,  after  this  method  of  treat- 


42  ON  NEUROKERATIN. 

ment  and  in  such  an  intermingled  mass  of  connective-tissue 
fibers,  any  nerve  fibers  showing  so  plainly  the  horny  sheaths 
and  the  spongy  network  of  neurokeratin  as  many  of  these 
do.  The  final  treatment  of  the  preparation  with  caustic 
soda  occasionally  causes  difficulty,  but  this  can  as  a  rule  be 
carried  on  so  easily  under  a  cover-glass  that  there  is  little 
danger  to  be  apprehended. 

There  remains  a  single  word  to  be  added  concerning 
the  peculiar  behavior  of  frog's  nerves  toward  pancreatic 
juice.  In  the  isolation  of  neurokeratin  from  such  nerves 
by  means  of  trypsin,  doubtless  many  have  obtained  unsatis- 
factory results,  since  in  this  case  the  connective  tissue  dis- 
appears and  the  nerve  fibers  fall  apart.  On  shaking  or  agi- 
tating such  a  preparation,  after  the  myeline  bodies  have 
been  removed,  the  greater  part  of  the  residue  from  the  tis- 
sue will  undoubtedly  be  broken  and  lost,  although  the 
splinters  of  neurokeratin  still  recognizable  will  not  be  de- 
prived of  their  characteristic  appearance  nor  fail  to  show 
their  insolubility  in  caustic  alkali.  This  difference  in  be- 
havior toward  trypsin  is,  however,  of  decided  advantage, 
for  it  facilitates  the  penetration  of  the  various  solvents  em- 
ployed, and  further  yields  the  nerve  fibers  isolated  in  a 
manner  fully  equal  to  the  result  obtained  by  gastric  diges- 
tion, bat  with  the  added  advantage  of  their  being  far  less 
deformed,  since  the  slow  decomposition  of  the  connective 
tissue  by  trypsin  is  not  accompanied  by  the  swelling  and 
shortening  of  the  nerves  produced  by  a  pepsin-acid  mixture. 
The  thick  medullated  nerves  from  the  head  of  certain  fishes, 
such  as  the  barbel,  behave  in  a  similar  manner  toward  tryp- 
sin ;  they  likewise  yield  an  insoluble  neurokeratin  similar 
to  the  above,  but  in  more  delicate  forms  and  often  very 
beautifully  arranged.  We  were  somewhat  surprised,  how- 
ever, on  comparing  the  behavior  of  the  epidermal  cells  from 
the  skin  of  the  head  and  from  the  fins  of  the  barbel  with 


ON   NEUROKERATIN.  43 

those  from  mammalian  epidermis  and  from  the  skin  shed 
by  frogs,  to  find  that  while  the  latter  withstood  the  ni">t 
vigorous  and  long-continued  action  of  gastric  juice,  the  epi- 
dermal cells  from  the  fish  were  wholly  decomposed  by  the 
same  treatment,  so  that  no  trace  of  them  could  be  found. 
FTkidklbkrg  and  New  Haven,  IS*'.). 


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On  neurokeratin. 


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